Involvement of cholecystokininergic systems in anxiety-induced hyperalgesia in male rats: behavioral and biochemical studies

Abstract

Keeping in mind the increased pain complaints reported in anxious or depressive patients, our goal was to investigate in rats the consequences of an experimentally provoked state of anxiety/depression on pain behavior and on its underlying mechanisms. We therefore used a model of social defeat consisting of a 30 min protected confrontation followed by a 15 min physical confrontation, repeated during 4 d, that elicited symptoms close to those observed in humans with anxiety or depression. Indeed, 5 d later, animals subjected to social-defeat confrontation were characterized by a decrease of sweet-water consumption and of body weight, and a hyperactivity of the hypothalamic-pituitary-adrenal axis, suggesting that the social-defeat procedure induced a prolonged state of anxiety. Rats subjected to the social-defeat procedure showed an enhanced nociceptive behavior to the subcutaneous administration of formalin, 5 d after the last confrontation session. Because chronic treatment with the established anxiolytic chlordiazepoxide (10 mg.kg(-1).d(-1)) prevented hyperalgesia, this strongly suggested that this experimental procedure might be a suitable animal model of "anxiety-induced hyperalgesia." Hyperalgesia associated with anxiety not only was related to a significant increase of CCKLM [cholecystokinin (CCK)-like material] in frontal cortex microdialysates but also was prevented by a CCK-B receptor antagonist [4-[[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2[[(tricyclo[3.3[12,17]dec-2-yloxy)-carbonyl]amino]-propyl]amino]-1-phenyethyl]amino]-4-oxo-[R-(R*, R*)]-butanoate N-methyl-D-glucamine (CI-988)] (2 mg/kg), strongly supporting the involvement of central CCKergic systems in these phenomena. Finally, combined treatments with CI-988 and morphine completely suppressed pain-related behavior, supporting the idea that the association of both compounds might represent a new therapeutic approach to reduce the increase of pain complaints highly prevalent among anxious or depressive patients.

Figure 1.

Long-term weight change induced by the social-defeat procedure. Daily body weight in nondefeated and defeated rats was measured before, during, and after the fourth days of conditioning sessions. Body weight measured on day 2 is the consequence of the first conditioning session. Each point is the mean ± SEM of data obtained in 10 defeated and 10 nondefeated intruders. *p < 0.002 and **p < 0.001 versus nondefeated intruders. Time course of sweet-water consumption in defeated and nondefeated animals is shown. One week before the beginning of the conditioning sessions (days –7 to 1) until 6 d after the end of these sessions (day 9), the animals had permanent access to two bottles (one of water and one containing 2% sucrose). Sucrose and water intakes were measured daily. The decrease of sweet-water consumption measured on day 2 is the consequence of the first conditioning session. Results are expressed as a percentage of control values (days –7 to 1) for each group. *p < 0.05, **p < 0.01, and ***p < 0.001 versus nondefeated intruders. D, Day.

Figure 2.

A, Time course of pain scores in defeated and nondefeated animals. Behavioral pain scores were recorded after injection of 2.5 or 5% formalin (50 μl) into the dorsal surface of the right hindpaw. Each point is the mean ± SEM of behavioral score obtained in 10–13 defeated and 9–10 nondefeated intruders. *p < 0.05, **p < 0.01, and ***p < 0.001 versus corresponding nondefeated intruders. B, Effects of 2.5 or 5% formalin injection on dialysate cortical levels of CCKLM in defeated or nondefeated intruders. Cortical CCKLM levels were measured in 30 min collected fractions (90 μl). Fraction 1 was the first fraction collected after a 90 min washout period after microdialysis probe insertion. Formalin (2.5 or 5%; 50 μl) was injected into the dorsal surface of the right hindpaw of defeated or nondefeated intruders, 8 min after the beginning of fraction 5 (arrow). Data are the means ± SEM of CCKLM contents in collected fractions (30 min each), expressed as percentages of basal values, taken as the mean of fractions 1–3. *p < 0.001 and **p < 0.0001 compared with basal values (100%). ^†p < 0.02, ^††p < 0.01, and ^†††p < 0.001 versus corresponding defeated intruders.

Figure 3.

A, Morphine effects on behavioral pain scores recorded after 2.5% formalin injection in defeated or nondefeated intruders. Morphine (4 mg/kg, s.c.) or saline was administered 40 min before injection of 2.5% formalin in defeated or nondefeated intruders. Each point is the mean ± SEM of behavioral score. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus defeated intruders injected with saline. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001, and ^††††p < 0.0001 versus nondefeated intruders injected with saline. a, p < 0.05; aa, p < 0.01 versus defeated intruders injected with morphine. B, Morphine effects on dialysate cortical CCKLM outflow in defeated and nondefeated intruders injected with formalin. Morphine (4 mg/kg, s.c.) or its vehicle (saline) was administered 40 min (i.e., 2 min before fraction 4; small arrow) before injection of 2.5% formalin (corresponding to 8 min after the beginning of fraction 5; large arrow) in defeated or nondefeated intruders. Data are the means ± SEM of CCKLM contents of collected fractions, expressed as percentages of basal values, taken as the mean of fractions 1–3. Fraction 1 was the first sample collected after a 90 min washout period after probe insertion. *p < 0.001 and **p < 0.0001 compared with basal values (100%). ^††p < 0.01 and ^†††p < 0.001 versus defeated intruders injected with saline. a, p < 0.0001 versus defeated intruders injected with saline.

Figure 4.

A, Effects of chronic treatment with chlordiazepoxide or CI-988 injection (with or without morphine) on behavioral pain scores recorded in intruders after 2.5% formalin injection. Chlordiazepoxide pumps or saline pumps were implanted subcutaneously on the day of microdialysis surgery for at least 4 d. On the ninth day, during the microdialysis experiment, morphine (4 mg/kg, s.c.), CI-988 (2 mg/kg, i.p.), or a combination of morphine plus CI-988 was administered 40 min before injection of 2.5% formalin. Each point is the mean ± SEM of the behavioral score. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus defeated intruders implanted with saline pump. ^†p < 0.05 and ^††p < 0.0001, defeated intruders implanted with chlordiazepoxide pump or injected with CI-988 in association with morphine versus corresponding values for defeated intruders(implanted with chlordiazepoxide pump alone or injected with CI-988 alone). B, Effects of chronic treatment with chlordiazepoxide (with or without morphine) on dialysate cortical CCKLM outflow in formalin-injected intruders. Formalin was injected 8 min after the beginning of fraction 5 (large arrow). Morphine (4 mg/kg, s.c.) was administered 40 min (i.e., 2 min before fraction 4; small arrow) before injection of 2.5% formalin in defeated intruders implanted with a chlordiazepoxide pump. Data are the means ± SEM of CCKLM contents in collected fractions, expressed as percentages of basal values, taken as the mean of fractions 1–3. Fraction 1 was the first sample collected after a 90 min washout period after probe insertion. *p < 0.001 and **p < 0.0001 compared with basal values (100%); ^†p < 0.05, ^††p < 0.01, and ^†††p < 0.001 versus corresponding values for defeated intruders implanted with saline pump.